Enzymatic preparation of D-glucose comprises a step for liquefying starch (liquefaction of gelatinized starch by alpha-amylase) and a step for saccharifying liquefied starch (saccharification of liquefied starch by glucoamylase). As corn starch is increasingly used as a raw material for the production of D-glucose in these days, alpha-amylase resistant starch (hereinafter referred to as "alpha-RS") which is hardly liquefied is troublesomely produced. Since alpha-RS is not hydrolyzed in the subsequent saccharifying step either, it makes filtration of the saccharified liquid difficult. Further, it reduces transparency of the liquid product so that a high quality product is not obtained, and the yield of D-glucose is decreased. Its disposal is also troublesome as it is hardly hydrolyzed.
An enzyme that hydrolyzes alpha-RS has not been known. There are known several methods for hydrolyzing alpha-RS to obtain a transparent product including a two-step method which comprises reheating the liquefied product to a temperature from 120.degree. to 140.degree. C., cooling to about 80.degree. C. and adding a liquefying enzyme to hydrolyze it, and a method comprising gelatinizing starch at a temperature higher than 100.degree. C. and liquefying it to suppress production of alpha-RS. However, the two step method requires a large amount of energy to gelatinize or liquefy starch and further special equipment since gelatinized starch has very high viscosity. On the contrary, in a one step method in which starch is saccharified at a temperature of from 25.degree. to 70.degree. C., the viscosity of the starch solution is low and the steps are simplified.
As a result of extensive study, it has now been found that specific strains belonging to genus Aspergillus produce enzymes which effectively hydrolyze starch including alpha-RS and can be used in the one step method for preparing D-glucose from starch.